Tenebrio antifreeze proteins

ABSTRACT

A novel class of thermal hysteresis (antifreeze) proteins (THP) that have up to 100 times the specific activity of fish antifreeze proteins has been isolated and purified from the mealworm beetle,  Tenebrio molitor.  Internal sequencing of the proteins, leading to cDNA cloning and production of the protein in bacteria has confirmed the identity and activity of the 8.4 to 10.7 kDa THP. They are novel Thr- and Cys-rich proteins composed largely of 12-amino-acid repeats of cys-thr-xaa-ser-xaa-xaa-cys-xaa-xaa-ala-xaa-thr. At a concentration of 55 μg/mL, the THP depressed the freezing point 1.6° C. below the melting point, and at a concentration of ˜1 mg/mL the THP or its variants can account for the 5.5° C. of thermal hysteresis found in  Tenebrio  larvae. The THP function by an adsorption-inhibition mechanism and produce oval-shaped ice crystals with curved prism faces.

CROSS REFERENCE TO RELATED APPLICATIONS

Not applicable.

BACKGROUND OF THE INVENTION

In modern times, refrigeration and, particularly, freezing have becomecommon and preferred means for storage of biological materials. Whilerefrigeration preserves some important properties of the samples, otherscontinue to deteriorate at a slow but significant rate. Frozen storagemay arrest most of this deterioration, but the combination of freezingand thawing introduces other changes which destroy other importantproperties.

In the modern world, frozen foods have become a mainstay of the humandiet. To ensure a high quality product, sufficient for the demandingconsumer's palate, frozen vegetables in particular, and frozen desserts,such as ice cream, have been the subject of extensive research by foodprocessors. It is now known that ice recrystallization can have asubstantial negative impact on the taste and texture of frozen foods.The advent of frost-free freezers has exacerbated this situation, whichhas been more traditionally associated with temperature fluctuationsduring transportation. After a relatively short period of time at otherthan sub-zero temperatures or even at sustained freezing temperatures,many frozen foods become less desirable, or worse, totally unsuitable,for human consumption.

While a variety of techniques have been implemented to mitigate thedamages associated with recrystallization, and limited success has beenattained, significant problems remain. Often, modifications to theprocessing of the frozen foods drastically affect their quality, color,flavor, and/or texture. Moreover, the additional processing can be veryexpensive and time consuming, rendering the techniques uneconomical.Similar difficulties have been associated with incorporating additivesto the foodstuffs.

For biologics, such as therapeutic drugs, blood plasma, mammalian cellsfor use in tissue culture, and the like, freezing can cause extensivedamage. For example, the freezing process itself kills most eukaryoticcells, and cells subjected to even one freezing and thawing cycleexhibit greatly reduced viability. Impaired function of living cells isalso prevalent in tissue cryopreservation, with concomitant drawbacksfor organ transplants. Similarly, frost or other freezing damage toplants presents a serious problem in agriculture. Finally, drugs canbecome ineffective, or even dangerous, if not maintained under requiredstrict temperature conditions.

Although the first description of protein-mediated thermal hysteresis(TH, as defined below) was noted in Tenebrio molitor hemolymphapproximately 30 years ago (Grimstone, et al, Philos. Trans. B 253:343(1968)), numerous attempts to purify these thermal hysteresis proteins(THP) failed to yield pure fractions with enough TH to account for thehemolymph activity (Grimstone, et al., (1968); Paterson & Duman, J. Exp.Zool. 210:361 (1979); Schneppenheim & Theede Comp. Biochem. Physiol.67B:561 (1980); Tomchaney, et at, Biochemistry 21:716 (1982); Paterson &Duman J. Exp. Zool. 219:381 (1982); and (Horwath, et al., Eur. J.Entomol. 93: 419 (1996)). Homogeneity of these proteins was not proven,and they differed in amino acid composition from each other and from thecompositions reported here.

There exists a need for new techniques and compositions suitable forimproving the preservation characteristics of organic materials at lowtemperatures, including storage of frozen foods and the viability ofbiologics. Ideally, these techniques and compositions will beinexpensive, yet completely safe and suitable for human consumption orin vivo therapeutic uses. There also exists a need for new techniquesand compositions suitable for depressing the freezing point orinhibiting freezing in non-organic systems, such as in deicingtreatments. The present invention fulfills these and other needs.

SUMMARY OF THE INVENTION

The common yellow mealworm beetle, Tenebrio molitor, is afreeze-tolerant pest of stored grains in temperate regions. Larvae areable to supercool to an average temperature of −12° C. (Johnston & Lee,Cryobiol. 27:562 (1990)). A second line of defense against freezing isthe thermal hysteresis (TH) activity of their hemolymph, which allowsthe insects to depress their freezing points in the presence of ice orice nucleators. This activity is quantified as the temperaturedifference (° C.) between the freezing and melting points of a solutioncontaining ice. Values for TH in Tenebrio hemolymph range from 1 to 10°C. according to the method of measurement and the conditions under whichthe insects are reared (Hansen & Baust, Biochim. Biophys. Acta 957:217(1988); and Patterson & Duman, J. Exp. Biol. 74:37 (1978)).

This invention provides for the nucleic acid molecules that encode theproteins responsible for the thermal hysteresis in Tenebrio larvae.Nucleic acid sequencing predicts a thermal hysteresis protein (THP)having at least greater than one repeat of a 12 contiguous amino acidmotif This repeating motif is rich in cysteine and threonine (SEQ IDNO:1). In addition to the repeating motif, the N-terminus of the classof THP of this invention is a 14 amino acid motif (SEQ ID NO:3).

In another embodiment, this invention provides for the recombinantproteins derived from the nucleic acids of this invention. The proteinis characterized as having a calculated molecular weight of between 7and 13 kDa, a pI of about 8 to 10 and a TH activity of greater thanabout 1.5° C. at 1 mg protein/mL.

The invention also provides for antibodies raised against the proteinsof this invention and antibodies that bind to the proteins of thisinvention. The invention provides for antibodies specificallyimmunoreactive under immunologically reactive conditions to anantifreeze protein comprising SEQ ID NO: 4. The invention also providesfor an antibody, specifically immunoreactive under immunologicallyreactive conditions, to an antifreeze protein comprising the proteinencoded by the nucleic acid of claim 1.

In a further embodiment of this invention, transformed yeast, bacteriaand other transgenic organisms are provided for. Many frozen foodstuffssuffer from formation of ice crystals due to sustained subfreezingtemperatures or repeated freeze-thaw cycles. The presence of the THP ofthis invention will provide for a longer shelf-life, making thesefoodstuffs more palatable. Transgenic animals and plants are envisionedas better surviving sub-freezing temperatures.

The invention provides for an organism into which, or into an ancestorthereof, an exogenous nucleic acid sequence which specificallyhybridizes under stringent conditions to SEQ ID NO: 2 or 5 or thenucleic acid of claim 1 has been introduced, and the organism translatesthe exogenous nucleic acid into an antifreeze protein. Also provided foris an organism with an exogenous nucleic acid sequence which istranslated into an antifreeze protein that is expressed externally fromthe organism. In a preferred embodiment, the organism is a fish. Infurther preferred embodiments, the organism is a fish is kept in asalt-water environment, or, the fish is a member of the familySalmonidae. In other preferred embodiments, the organism can be a plant,a fungus, a yeast or a bacteria. In another embodiment, if the organismis a yeast, it can be selected from the group consisting of Torulopsisholmil, Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyceslactis, and Candida pseudotropicalis. In another embodiment, if theorganism is a bacterium, it can be selected from the group consisting ofEscherichia coli, Streptococcus cremoris, Streptococcus lactis,Streptococcus thermophilus, Leuconostoc citrovorum, Leuconostocmesenteroides, Lactobacillus acidophilus, Lactobacillus lactis,Bifidobacterium bifidum, Bifidobacterin breve, and Bifidobacteriumlongum. Plants transformed with THP sequences can include grapes,oilseed plants such as canola, grains, citrus and sugar cane.

The invention provides for a method for decreasing the freezing point ofan aqueous solution involving the addition of an antifreeze protein tothe aqueous solution. In a preferred embodiment, the method involves theaddition of the antifreeze protein encoded by the nucleic acid of claim1 to the aqueous solution. In other preferred embodiments, the aqueoussolution is applied to an organism; the antifreeze protein is producedby recombinant means; the antifreeze protein can specifically bind tothe antibody of claim 13 or claim 14; the antifreeze protein is selectedfrom the group consisting of YL-1, YL-2, YL-3 and YL-4; and/or, theantifreeze protein is encoded by a nucleic acid molecule whichspecifically hybridizes to the nucleic acid of SEQ ID NO:2 or 5.

In addition, it is contemplated that the addition of the THP of thisinvention to aqueous solutions may better preserve organs and otherbiologicals in transit.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification, the figures and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chromatogram of diluted Tenebrio hemolymph loaded onto anS-100 SEPHACRYL® (Pharmacia) column (92 cm×1.6 cm) and eluted withhemolymph buffer without phenylthiocarbamide (see text).

FIG. 2 is a chromatogram of selected active fractions from gel exclusionchromatography which were combined and chromatographed by reversed-phaseHPLC on a C18 analytical column (Vidac), using a gradient of 0.4%acetonitrile/min in 0.05% trifluoroacetic acid.

FIG. 3 is a 15% SDS-PAGE of reverse-phase HPLC fractions which werelyophilized and resuspended in 50 μL of 0.1 M NH₄HCO₃ (pH 8.0). The gelwas stained using the Silver Stain Plus Kit (Bio-Rad). Approximatemolecular weights were determined by MALDI mass spectrometry.

FIG. 4 is a graph indicating the hyperbolic nature of the thermalhysteresis activity of Tenebrio hemolymph.

FIGS. 5 I-IV, are micro-photographs of ice crystals grown in thepresence of Tenebrio THP (I and II) and fish antifreeze proteins (IIIand IV).

FIG. 6 is an alignment chart of recombinant isoforms of THP. Thepositions in which the nucleotide is conserved in all cDNA sequences aremarked by an asterisk (*). The complete amino acid sequence is indicatedonly for YL-1. Residues of other isoforms which are identical to thosefound in YL-1 are indicated by a period (.). Differences are shown wherefound. Gaps in both the cDNA and protein sequences are indicated bydashes (------).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to isolated nucleic acid sequences encoding anovel class of antifreeze protein (THP). The procedure for obtainingnatural THP genes generally involves constructing or obtaining a genelibrary from Tenebrio molitor, detecting and isolating the desired gene,cloning it, and expressing it in a suitable host cell and then purifyingthe expressed protein. The natural protein can be used withoutmodification or it can be modified in a variety of ways withoutaffecting its TH activity.

The amino acid compositions of the purified proteins and the deducedmature sequences are very similar. The THPs are particularly rich in Cys(18-19%) and Thr (20-26%) and deficient in several hydrophobic aminoacids, notably Leu and Ile. The overall hydrophilicity is approximately55% (Wishard, et al., Comput. Appl. Biosci. 10:121 (1994)), which ismuch higher than that found in fish antifreeze proteins (AFPs)(Sonnichsen, et al., Prot. Sci. 4:460 (1995)).

The primary structure of the mature THP is very unusual (FIG. 4) and isnot similar to any other known sequence. The first 21 amino acidscontain 6 Cys spaced at irregular intervals (Cx₅Cx₂Cx₃Cx₂Cx₂C; SEQ IDNO:3), and this sequence overlaps with the first of a series of12-amino-acid repeats that continue until the end of the protein. Cys isrepeated at 6-residue intervals throughout this region, which has theconsensus sequence CTxSxxCxxAxT (SEQ ID NO: 1). The N-terminal Cysspacing has some elements in common with zinc-binding motifs (Klug &Schwabe, FASEB J. 9:597 (1995)). However, extensive dialysis against 10mM EDTA or 10 mM phenanthroline, and the subsequent addition of 2 mMZnCl₂ (or 2 mM CaCl₂) to chelator-free preparations incubated for 1 h at22° C. does not affect activity, suggesting that there is no role fordivalent metal ions in TH activity. At least some of the Cys residuesare involved in disulfide bridges because all activity is lost onincubation with 10 mM dithiothreitol at 37° C. for 20 min, whereas noactivity is lost under the same conditions in the absence of reducingagent. There is no effect of N-ethylmaleimide on TH activity, whichsuggests that if free Cys are present they can be modified without lossof activity.

The differences in length between the THP variants represent multiplesof the 12-amino-acid repeat, suggesting that each repeat may form afunctional domain. Although the THP repeats are short, they may foldindependently to form a chain, which could explain the discrepancybetween the actual and apparent molecular weights (see, infra) of theproteins as being due to asymmetry.

There is no structural similarity between Tenebrio THPs and fish AFPs.Fish type II AFP, which contains 10 Cys, does not display any repetitivestructure or regularity in Cys spacing. Fish type I AFP and theantifreeze glycoprotein are built up of repetitive elements, but neithercontains Cys (Davies & Hew, FASEB J. 4:2460 (1990)). It is, however,intriguing that the most abundant residue in Tenebrio THPs, Thr, is alsothe amino acid thought to play a central role in binding fish AFPs typesI and III to the ice surface (Wen & Laursen J. Biol. Chem. 267:14102(1992); and Chao. et al., Prot. Sci. 3:1760 (1994)).

However, like fish antifreeze proteins, Tenebrio THP appears to act byan adsorption-inhibition mechanism. Ice crystals in the presence ofsufficient THP to produce about 1° C. of thermal hysteresis, stopgrowing until the non-equilibrium freezing point is exceeded. At thatpoint, ice crystals burst forth from the crystal nucleator to form asolid mass of ice along the a-axis. At low thermal hysteresisactivities, the ice fronts are broad and smooth. However, at highthermal hysteresis values, the ice fronts are no longer smooth. Incontrast, once the freezing point is exceeded in the presence of fishAFPs, myriad ice spicules burst out along and parallel to the c-axis.

In addition, similar to fish AFP (DeVries, Annu. Rev. Physiol. 45:245(1983), the relationship between TH activity and THP concentration ishyperbolic. However, the ice crystals formed in the presence of THP areunusual in that they have a pronounced curvature of their surfaces. Incontrast, ice crystals generated by fish AFP Types I and III arehexagonal bipyramids with flat, well-defined facets.

The purified, expressed THP protein can be directly added to an aqueoussolution to depress the freezing point or in another embodiment,transformed organisms that express the antifreeze proteins can be addedto items which will be stored frozen, such as frozen desserts. In yetanother embodiment, the transformed organisms, e.g., fish, plants andyeast, need not express the THP proteins extracellularly but thepresence of a THP gene and intracellular protein confers to them theincreased ability to survive freezing temperatures. For example, atransformed organism can be a saltwater fish. Transformed salt-waterfish can include members of the family Salmonidae, halibut, sablefish orany edible saltwater species not having any or sufficient levels ofantifreeze proteins. Plants transformed with THP sequences can includegrapes, oilseed plants such as canola, grains, citrus and sugar cane.

All publications, patents and patent applications cited herein arehereby expressly incorporated by reference for all purposes.

I. DEFINITIONS

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds to and recognizes an analyte (antigen). Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(I)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂ a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)l by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, FUNDAMENTAL IMMUNOLOGY, 3RD ED., W. E. Paul, ed.,Raven Press, N.Y. (1993)). While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv).

An “antifreeze protein antibody” is an antibody or antibody fragmentthat specifically binds an antifreeze protein of this invention or asubsequence thereof.

The term “antifreeze protein” refers to a protein found in the bodyfluids of some poikilothermic organisms, such as the Tenebrio molitormealworm and plants, which have the commonly known property that theyreduced non-colligatively the freezing point of water. Antifreezeproteins are also known as “thermal hysteresis proteins” (THPs). As usedherein, “antifreeze proteins” or “THPs” includes chemically synthesizedand recombinantly produced polypeptides having a protein sequence withsubstantial similarity to a naturally occurring antifreeze protein andretaining the properties of a natural antifreeze protein.

The phrase “consensus sequence” refers to a nucleic acid or amino acidsequence in which each position represents the residue or base mostoften found when many actual sequences are compared. SEQ ID NO:1 is aconsensus sequence of a repeating motif found in the proteins of theinstant invention

The phrase “conserved amino acids” refers to common amino acid residuesin two or more appropriately aligned THP-motif sequences. The conservedamino acid sequences can be intrapeptide or interpeptide. For example,in the THPs of the present invention, the amino acid motifs may consistof 5 out of 6 conserved amino acids, or in other words, 5 out of 6 aminoacid residues of any particular motif will be common with 5 out of 6amino acids of the motif either within a THP amino acid sequence orwithin two or more THP amino acid sequences.

The term “contiguous amino acid motif” refers to a repeating pattern ofamino acids present in a polypeptide or protein. The amino acids in eachrepeat do not have to be the same but there should be a pattern commonto all. For example, in the class of proteins of the present invention,the repeating amino acid motif; cys-thr-xaa-ser-xaa-xaacys-xaa-xaa-ala-xaa-thr, where xaa is any amino acid, is present.

The term “crustacean” refers to the common definition of the word, i.e.,a chiefly aquatic arthropod of the class Crustacea, including lobsters,shrimps, crabs and barnacles.

The term “decreasing the freezing point of an aqueous solution” refersto lowering the temperature of an aqueous solution at which ice crystalsform and grow. The decrease in freezing point depends both on the agentused to decrease tile freezing point and its concentration in theaqueous solution. The freezing point depression increases as theantifreeze component is added to the aqueous solution, until a maximumdepression is observed at a characteristic concentration. Furtheraddition of antifreeze chemicals, such as ethylene glycol, to aqueoussolutions will either result in insolubility of the antifreezecomposition or serve to increase the freezing point of the mixture. Onthe other hand, the increase in thermal hysteresis with increased THPconcentration is not linear but hyperbolic. The incremental increases inTH that result from unit increases in THP concentration become smallerand smaller as the saturation point of the THP solubility is approached.

The freezing point of a solution with a THP protein is defined as thetemperature at which the sample being measured, which contains an icecrystal nucleator, becomes a solid mass of ice. The ice crystals canform spontaneously or expand from an ice nucleator. Spontaneousformation of a solid mass of ice without a nucleator is typically termedthe “supercooling ability” of the THP. Because of the absence of an icenucleator to initiate the ice formation process, supercooling can occurat much lower temperatures.

In addition to inspecting visually ice crystal formation, a thermalhysteresis assay can measure the difference between the freezing andmelting points of a solution. The melting point of a solution is thetemperature at which there is only one ice crystal left in a solution(see, infra, for a more complete description of TH activity).

The phrase “expressed externally” in the context of a recombinantprotein refers to the ability of the transformed cell to synthesize anddirect the protein into the extracellular matrix. The extracellularmatrix can be the interstitial space between cells in a multicellularorganism, bacterial broth or tissue culture medium. With bacteria,external expression can also include expression of the recombinantprotein into the periplasm of the bacteria. External expression can beby any means, e.g., secretory and transport vesicles, expression as amembrane protein, expression of a cleavable signal peptide, etc.

The terms “homology,” “sequence identity” and “sequence similarity” inthe context of this invention mean that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 40 percent sequence identity, preferably atleast 50 percent sequence identity, and most preferably at least 60percent sequence identity. “Percentage amino acid sequence identity”refers to a comparison of the amino acid sequences of two polypeptideswhich, when optimally aligned, have approximately the designatedpercentage of the same amino acids. For example, “60% sequence identity”and “60% homology” refer to a comparison of the amino acid sequences oftwo polypeptides which, when optimally aligned, have 60% amino acididentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions. For example, the substitutionof amino acids having similar chemical properties such as charge orpolarity is not likely to affect the properties of a protein. Examplesinclude glutamine for asparagine or glutamic acid for aspartic acid.

The term “immunologically reactive conditions” refers to an environmentin which antibodies can bind to antigens. Typically, this is animmunological binding assay.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state, although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis (PAGE) or high performance liquid chromatography (HPLC).A protein which is the predominant species present in a preparation issubstantially purified.

In particular, an isolated THP gene is separated from open readingframes which naturally flank the gene and encode proteins other than theTHP. The term “purified” denotes that a nucleic acid or protein givesrise to essentially one band in an electrophoretic gel. Particularly, itmeans that the nucleic acid or protein is at least 85% pure, morepreferably at least 95% pure, and most preferably at least 99% pure.

The term “nucleic acid molecule” or “nucleic acid sequence” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides which have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g. degenerate codon substitutions) and complementarysequences and as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes 8:91-98 (1994)). The term nucleic acid is used interchangeablywith gene, cDNA, and mRNA encoded by a gene.

The phrase “exogenous nucleic acid” generally denotes a nucleic acidthat has been isolated, cloned and ligated or chemically synthesized andligated to a nucleic acid with which it is not combined in nature,and/or introduced into and/or expressed in a cell or cellularenvironment other than the cell or cellular environment in which saidnucleic acid or protein may typically be found in nature. The termencompasses both nucleic acids originally obtained from a differentorganism or cell type than the cell type in which it is expressed, andalso nucleic acids that are obtained from the same cell line as the cellline in which it is expressed.

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidwhich contains sequence information for a structural RNA such as rRNA,or tRNA, or for mRNA which encodes the primary amino acid sequence of aspecific protein or peptide, or a binding site for a trans-actingregulatory agent. This phrase specifically encompasses degenerate codons(i.e., different codons which encode a single amino acid) of the nativesequence or sequences which may be introduced to conform with codonpreference in a specific host cell.

The term “recombinant means” refers to techniques where proteins areisolated, wherein the cDNA sequence coding the protein is identified andinserted into an expression vector. The vector is then introduced into acell and the cell expresses the protein. Recombinant means alsoencompasses the ligation of coding or promoter DNA from differentsources into one vector for expression of a fusion protein, constitutiveexpression of a protein, or inducible expression of a protein.

The phrase “specifically or selectively binds to an antibody” or“specifically immunoreactive with”, when referring to a protein orpeptide, refers to a binding reaction which is determinative of thepresence of the protein in the presence of a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein and donot bind in a significant amount to other proteins present in thesample. Specific binding to an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example. antibodies raised against the THP ofthis invention or to the partially encoded sequence depicted in SEQ IDNO: 4 can be selected to specifically immunoreact with full lengthprotein and not with other proteins except perhaps to polymorphicvariants. As described below, a variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See Harlow & Lane (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York (“Harlow & Lane”), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity. Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

The term “specifically hybridizing” refers to a nucleic acid probe thathybridizes, duplexes or binds to a particular target DNA or RNA sequencewhen the target sequences are present in a preparation of total cellularDNA or RNA. “Complementary” or “target” nucleic acid sequences refer tothose nucleic acid sequences which selectively hybridize to a nucleicacid probe. Proper annealing conditions depend, for example, upon aprobe's length, base composition, and the number of mismatches and theirposition on the probe, and must often be determined empirically. Fordiscussions of nucleic acid probe design and annealing conditions, see,for example, Sambrook or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubelet al., ed. Greene Publishing and Wiley-Interscience, New York (1987)(“Ausubel”).

“Stringent hybridization” and “stringent hybridization wash conditions”in the context of nucleic acid hybridization experiments such asSouthern and northern hybridizations are sequence dependent, and aredifferent under different experimental parameters. An extensive guide tothe hybridization of nucleic acids is found in Tijssen (1993) LABORATORYTECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY—HYBRIDIZATION WITHNUCLEIC ACID PROBES part I chapter 2 “Overview Of Principles OfHybridization And The Strategy Of Nucleic Acid Probe Assays”, Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature, under defined ionic strength and pH, atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleic acids which have more than100 complementary residues on a filter in a Southern or northern blot is50% formamide with heparin at 42° C., with the hybridization beingcarried out overnight. An example of highly stringent wash conditions is0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook,supra for a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratio of2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

The term “thermal hysteresis activity” or “TH activity” refers to theability to alter the temperature difference (° C.) between the freezingand melting points of a solution containing ice. Preferably, TH activityis be measured by observation of ice crystal formation in a nanoliterosmometer following the procedure set forth in Lawson & Semler, Proc.Nat'l Acad. Sci. USA 88:9919 (1991). Alternatively, TH activity can bedetermined according to the method described in deVries, METHODS INENZYMOLOGY, VOL. 127, Packer (ed.), Academic Press, New York (1986) or avariation thereof.

For example, in the present invention, the TH activity of the nativeTenebrio molitor THP from hemolymph at approximately 1 mg/mL is greaterthan approximately 5° C. The TH activity of the recombinant THP of thisinvention at approximately 1 mg/mL is about 5° C. or less. Morepreferably, the TH activity is lower than about 3° C. Most preferably,the TH activity is between 1.5 and 3° C. It is hypothesized that thedifferences between the TH activity of the native THP and therecombinant THP is due to imperfect folding of at least a portion of theTHP of the bacteria-derived recombinant proteins. When properly folded,it is expected that the TH activities of the recombinant THP will besimilar to that of the native THP.

II. NUCLEIC ACIDS ENCODING THP

A. General Techniques

The nucleic acid compositions of this invention, whether RNA, cDNA,genomic DNA, or a hybrid of genetic recombinations, may be isolated fromnatural sources or may be synthesized in vitro. The nucleic acidsclaimed may be present in transformed cells, in a transformed celllysate, or in a partially purified or substantially pure form.

Techniques for nucleic acid manipulation of genes encoding theantifreeze protein such as generating libraries, subcloning intoexpression vectors, labeling probes, DNA hybridization, and the like aredescribed generally in Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL (2ND ED.), Vols. 1-3, Cold Spring, Harbor Laboratory, N.Y. (1989)(“Sambrook”), which is incorporated herein by reference.

Nucleic acids and proteins are detected and quantified herein by any ofa number of means well known to those of skill in the art. These includeanalytical biochemical methods such as spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, and various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.The detection of nucleic acids proceeds by well known methods such asSouthern analysis, northern analysis, gel electrophoresis, PCR®,radiolabeling, scintillation counting, and affinity chromatography.

B. Isolation of Nucleic Acids Encoding THP

Methods of isolating total DNA or mRNA are well known to those of skillin the art. For example, methods of isolation and purification ofnucleic acids are described in detail in Chapter 3 of LABORATORYTECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITHNUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, P.Tijssen, ed. Elsevier, N.Y. (1993) (“Tijssen”).

1. Preparation and Screening of DNA Libraries

There are numerous methods for isolating the DNA sequences encoding theantifreeze protein of this invention. For example, DNA may be isolatedfrom a genomic or cDNA library using labeled oligonucleotide probeshaving sequences complementary to the sequences or subsequencesdisclosed herein (SEQ ID NOs:2 or 5). Such probes can be used directlyin hybridization assays to isolate DNA encoding THP isoforms.Alternatively probes can be designed for use in amplification techniquessuch as PCR, and DNA encoding THP may be isolated by using methods suchas PCR (see infra).

To prepare a cDNA library, mRNA is isolated from Tenebrio larvae(Carolina Biological Supply, or a local pet or bait shop) or fromembryonic, larval or possibly adult cells grown in tissue culture. cDNAis reverse transcribed from the mRNA according to procedures well knownin the art and inserted into vectors. The vectors are transfected into arecombinant host for propagation, screening, and cloning. Methods formaking and screening cDNA libraries are well known. See Gubler &Hoffman, Gene 25:263 (1983) and Sambrook, et al.

To make a genomic library, total DNA is extracted from the cells of theinsect by well-known methods (see Sambrook, et al.) and thenmechanically sheared or enzymatically digested to yield DNA fragments(e.g., of about 12-20 kb). The fragments are then separated by gradientcentrifugation from undesired sizes and are inserted in bacteriophage-λor other vectors. These vectors (e.g., phage) are packaged in vitro, asdescribed in Sambrook, et al. Recombinant phage are analyzed by plaquehybridization as described in Benton & Davis, Science, 196: 180 (1977).Colony hybridization is carried out as generally described in Grunstein,et al., Proc. Natl. Acad. Sci. USA., 72:3961 (1975).

DNA encoding THP is identified in either cDNA or genomic libraries bythe ability to hybridize with nucleic acid probes, for example SEQ IDNO:2 or 5. Once identified, these DNA regions are isolated by standardmethods familiar to those of skill in the art. Alternatively, RNAencoding antifreeze protein may be identified by its ability tohybridize to nucleic acid probes in northern blots. See, Sambrook, etal.

Oligonucleotides for use as probes are chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage & Carruthers using an automated synthesizer, as described inNeedham-VanDevanter, et al., Nucleic Acids Res. 12:6159 (1984).Purification of oligonucleotides is by either native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson &Regnier, J. Chrom. 255:137-149 (1983). The sequence of the syntheticoligonucleotide can be verified using the chemical degradation method ofMaxam & Gilbert, Methods in Enzymology, 65:499 (1980).

Other methods known to those of skill in the art may also be used toisolate DNA encoding the antifreeze protein. See Sambrook and Ausubelfor descriptions of other techniques that can be utilized for theisolation of DNA encoding specific protein molecules.

2. Amplification of Nucleic Acids Encoding THP

Frequently, it is desirable to amplify the nucleic acid sample prior tohybridization and subsequent subcloning. One of skill in the art willappreciate that whatever amplification method is used, if a quantitativeresult is desired, care must be taken to use a method that maintains orcontrols for the relative frequencies of the amplified nucleic acids.Suitable amplification methods include, but are not limited to:polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS ANDAPPLICATIONS. Innis, et al., Academic Press, Inc. N.Y., (1990)(“Innis”)), ligase chain reaction (LCR) (see Wu and Wallace, Genomics,4:560 (1989) (“Wu”), Landegren et al., Science, 241:1077 (1988)(“Landegren”) and Barringer et al., Gene 89:117 (1990) (“Barringer”);transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA,86:1173 (1989) (“Kwoh”)); and, self-sustained sequence replication(Guatelli. et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)(“Guatelli”)).

All of the above methods can be used to prepare DNA encoding antifreezeprotein. In PCR techniques, oligonucleotide primers complementary to thetwo borders of the DNA region to be amplified are synthesized. Thepolymerase chain reaction is then carried out using the two primers(see, Innis). In the instant invention, because of the presence ofrepetitive motifs, the length of the THP subsequence encoded by theamplified product will depend on the template used. Because the N and Ctermini are unique (i.e., different nucleotide sequences from therepetitive motif), to amplify the full-length THP encoding sequence, theprimers of SEQ ID NOs: 6 and 7 can be used.

PCR can be used in a variety of protocols to isolate nucleic acidsencoding partial sequences of THP. In these protocols, appropriateprimers and probes for amplifying DNA encoding partial sequences of THPsare generated from analysis of the DNA sequences listed herein. Forexample, the oligonucleotides of SEQ ID NOs:6, 7, 8 and 9 can be used ina PCR protocol to amplify regions of DNA which encodes THPs. Once suchregions are PCR-amplified, they can be sequenced and labeledoligonucleotide probes can be prepared from the sequence obtained. Theseprobes can then be used to isolate DNA encoding the complete THP fromDNA libraries.

SEQ ID NOs:2 and 5 represent isoforms of naturally occurring Tenebriomolitor THP cDNA. They are not complete DNA gene sequences. However, thepartial antifreeze nucleic acid sequence of SEQ ID NO:2 or 5 can becompleted according to standard methods well known to those of skill inthe art. A preferred approach for DNA isolation is RACE. Briefly, thistechnique involves using PCR to amplify a cDNA sequence using a random5′ primer and a defined 3′ primer (5′ RACE) or a random 3′ primer and adefined 5′ primer (3′ RACE). The amplified sequence is then subclonedinto a vector where it is then sequenced using standard techniques. TheRACE method is well known to those of skill in the art and kits toperform RACE are commercially available (e.g. 5′ RACE System, GIBCO BRL,Grand Island, N.Y., USA).

3. Cloning of THP-encoding Inserts Into Bacteria

As described above, to prepare and screen genomic DNA total DNA isfragmented and inserted into bacteriophage. Once inserts containingantifreeze nucleic acid sequence of interest have been identified (byPCR, hybridization or the like), they are excised out of λ phage vectorsand inserted into bacterial vectors for expansion. Typically, suitablebacterial vectors are known to practitioners in the art and arecommercially available. The most suitable vector may depend on thebacteria to be used, the size of the insert, the method of detection ofbacteria which contain the insert of interest and the preference of thepractitioner.

To simplify identification of colonies of bacteria transformed withvectors containing the inserts, many vectors have restriction enzymesites or other splicing sites located within a coding sequence for anenzyme, in particular, β-galactosidase. If an insert has successfullybeen inserted into the vector at the restriction or splicing sites, theencoded enzyme is inactivated. After transformation of the bacteria withthe vector (see, infra), colonies grown in the presence of isopropylβ-D-thiogalactoside (IPTG) (a substrate for β-galactosidase) appearwhite, while the colonies derived from a bacteria which did notincorporate the insert appear blue. Thus, if the frequency of ligationof the insert into the vector was low, one can pick the few coloniesthat contain inserts over the many that will not.

The vectors are then introduced into variants of Escherichia coli.Methods used to introduce foreign DNA into bacterial cells are known tothose of skill in the art, but the most frequently used areelectroporation and heat shock of competent cells. Most typically,competent E. coli are provided commercially (for example fromInvitrogen, San Diego, Calif.). Alternatively, the bacteria can be madecompetent to take up foreign DNA by techniques well known in the art(see, Sambrook, et al.). To introduce the vectors containing the insertof interest into bacteria, the competent bacteria undergo a heat shockprocess. Briefly, the bacteria are held in an ice water bath and afterthe DNA has been added, the temperature of the bacteria is raised to40-50° C., preferably 42° C. The bacteria are returned to the ice bathand then cultured. For a more detailed description of introducing DNAinto bacteria see Sambrook, et al., which is incorporated by referenceherein.

The bacterial cultures are grown and then plated out on agar. Typically,the vector encodes a gene which confers resistance to an antibiotic tothe transformed bacteria. Therefore, bacteria which have taken up thevector survive and form colonies on agar plates permeated with theantibiotic. The surviving colonies can be used to incubate broth andexpanded into large cultures of transformed bacteria.

Plasmids and other vectors can be purified from bacterial lysates bymethods well known in the art. Many commercial suppliers sell kits forpurifying small circularized DNA (plasmids) from total bacterial DNA.These kits are easy to use and, by following the manufacturer'sinstructions, the yield is usually quite high.

4. Sequencing of THP DNA

Sequencing of newly isolated DNA will identify and characterize THPnucleic acid of the invention, this nucleic acid encoding THP species orallelic variations, the antifreeze proteins of the invention. A proteincan be considered a THP protein isoform if it has at least 60% aminoacid sequence identity to the Tenebrio molitor THP identified by SEQ IDNO:6 and SEQ ID NO:7.

THP coding sequences can be sequenced while they are still present asinserts in vectors or as inserts released and isolated from the vectors.THP-encoding inserts can be released from the vectors by restrictionenzymes or amplified by PCR. For sequencing of the inserts to discoverwhich inserts contain full length antifreeze coding sequences, primersbased on the N- or C-terminus, such as those of SEQ ID NO:8 and SEQ IDNO:9, or insertion points in the original λ-phage, can be used asprimers. Most preferably, additional primers are synthesized to providefor overlapping sequences. Typically, dideoxy sequencing is done(Sequenase®, U.S. Biochemical). However, other kits and methods areavailable and known to those of skill in the art.

As an alternative to the bacterial cloning described above, the positiveλ-phage plaques from the cDNA library can be subjected to in vivoexcision with helper phage, for example R408 (Stratagene). The doublestranded DNA can then be purified and sequenced or amplified by PCRusing primers derived from the λ-phage.

C. Nucleic Acid Hybridization Techniques

The hybridization techniques disclosed herein can be utilized toidentify, isolate and characterize genes and gene products (i.e., mRNA)encoding for the antifreeze proteins of the invention, includingdifferent THP species and allelic variations.

A variety of methods for specific DNA and RNA measurement using nucleicacid hybridization techniques are known to those of skill in the art.See NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH, Ed. Hames, B. D.and Higgins, S. J., IRL Press, 1985; Gall & Pardue, Proc. Natl. Acad.Sci., U.S.A. 63:378 (1969); John, et al., Nature 223:582 (1969); andSambrook, et al. The selection of a hybridization format is notcritical.

For example, one method for evaluating the presence or absence of DNAencoding antifreeze protein in a sample involves a Southern transfer.Briefly, digested genomic DNA is run on agarose slab gels in buffer andtransferred to membranes. Hybridization is carried out using nucleicacid probes. For the THP of this invention, the nucleic acid probes canbe designed based on conserved nucleic acid sequences amongst the classof proteins. Preferably nucleic acid probes are 20 bases or longer inlength. (See Sambrook, et al. for methods of selecting nucleic acidprobe sequences for use in nucleic acid hybridization.) In the instantinvention, preferable probes would be the sequences identified as SEQ IDNO:2 or 5 or the entire coding region of any of the isoforms.Visualization of the hybridized portions allows the qualitativedetermination of the presence or absence of DNA encoding antifreezeprotein.

Similarly, a northern transfer may be used for the detection of mRNAencoding antifreeze protein. In brief, mRNA is isolated from a givencell sample using one of a variety of extraction methods such as an acidguanidinium-phenol-chloroform. The mRNA is then electrophoresed toseparate the mRNA species and the mRNA is transferred from the gel to amembrane. As with the Southern transfers, labeled probes are used toidentify the presence or absence of THP mRNAs.

Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a“capture” nucleic acid covalently immobilized to a solid support and alabeled “signal” nucleic acid in solution. The clinical sample willprovide the target nucleic acid. The “capture” nucleic acid and “signal”nucleic acid probe hybridize with the target nucleic acid to form a“sandwich” hybridization complex. To be effective, the signal nucleicacid cannot hybridize with the capture nucleic acid.

Typically, labeled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labeled by any one of several methods typically used to detect thepresence of hybridized polynucleotides. The most common method ofdetection is the use of autoradiography or autofluorography with ³H,¹²⁵I, ³⁵S, ¹⁴C, or ³²P-labeled probes or the like. Other labels includeligands which bind to labeled antibodies, fluorophores, chemiluminescentagents, enzymes, and antibodies which can serve as specific binding pairmembers for a labeled ligand. Detection would then depend on the labelused.

Detection of a hybridization complex may require the binding of a signalgenerating complex to a duplex of target and probe polynucleotides ornucleic acids. Typically, such binding occurs through ligand andanti-ligand interactions as between a ligand-conjugated probe and ananti-ligand conjugated with a signal.

The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to tileantibodies or, in some cases, by attachment to a radioactive label (see,Tijssen).

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including PCR, LCR, Qβ-replicase amplificationand other RNA polymerase mediated techniques (e.g., NASBA™) are found inBerger, Sambrook, and Ausubel, as well as Mullis et al., U.S. Pat. No.4,683,202; Arnheim & Levinson, C&EN 36 (1990); Lomell et al., J. Clin.Chem., 35:1826 (1989); Van Brunt, Biotechnology 8:291-294 (1990); Wu &Wallace, Gene 4:560 (1989); Sooknanan & Malek, Biotechnology 13:563(1995); Innis; Kwoh; Guatelli; Landegren; and Barringer. Improvedmethods of cloning in vitro amplified nucleic acids are described inWallace et al., U.S. Pat. No. 5,426,039. Other methods recentlydescribed in the art are the nucleic acid sequence based amplification(NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicase systems. Thesesystems can be used to directly identify mutants where the PCR or LCRprimers are designed to be extended or ligated only when a selectsequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation.

Oligonucleotides for use as probes, e.g., in in vitro amplificationmethods, as gene probes in diagnostic methods, or as inhibitorcomponents (see below) are typically synthesized chemically according tothe solid phase phosphoramidite triester method described by Beaucageand Caruthers, e.g., using an automated synthesizer, as described inNeedham-VanDevanter. Purification of oligonucleotides, where necessary,is typically performed by native acrylamide gel electrophoresis or byanion-exchange HPLC as described in Pearson & Regnier. The sequence ofthe synthetic oligonucleotides can be verified using the chemicaldegradation method of Maxam & Gilbert.

It will be appreciated that nucleic acid hybridization assays can alsobe performed in an array-based format. In this approach, arrays bearing,a multiplicity of different “probe” nucleic acids are hybridized againsta target nucleic acid. In this manner a large number of differenthybridization reactions can be run essentially “in parallel”. Thisprovides rapid, essentially simultaneous, evaluation of a wide number ofreactants. Methods of performing hybridization reactions in array basedformats are well known to those of skill in the art (see, e.g., Jackson,et al., Nature Biotechnology 14:1685 (1996), and Chee, et al., Science274:610 (1995)).

An alternative means for determining the level of expression of a geneencoding a protein in situ hybridization. In situ hybridization assaysare well known and are generally described in Angerer, et al., MethodsEnzymol., 152:649 (1987). In an in situ hybridization assay, cells ortissues are fixed to a solid support, typically a glass slide. If DNA isto be probed, the cells are denatured with heat or alkali. The cells arethen contacted with a hybridization solution at a moderate temperatureto permit annealing of labeled probes specific to the nucleic acidsequence encoding the protein. The probes are preferably labeled withradioisotopes or fluorescent reporters.

D. Expression of THP

After the coding region of an antifreeze protein gene has beenidentified, the expression of natural or synthetic antifreeze-encodingnucleic acids can be achieved by operably linking the coding region ofan antifreeze protein gene to a promoter (which is either constitutiveor inducible), incorporating the construct into an expression vector,and introducing the vector into a suitable host cell. Typical vectorscontain transcription and translation terminators, transcription andtranslation initiation sequences, and promoters useful for regulation ofthe expression of the particular nucleic acid. The vectors optionallycomprise generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of thecassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors)and selection markers for both prokaryotic and eukaryotic systems.Vectors are suitable for replication and integration in prokaryotes,eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979);Roberts, et al., Nature 328:731 (1987); Berger & Kimmel, GUIDE TOMOLECULAR CLONING TECHNIQUES, METHODS IN ENZYMOLOGY, Vol 152, AcademicPress, Inc., San Diego, Calif. (“Berger”); Schneider, et al., ProteinExpr. Purif. 6435:10 (1995); Sambrook and Ausubel. Product informationfrom manufacturers of biological reagents and experimental equipmentalso provide information useful in known biological methods. Suchmanufacturers include the SIGMA chemical company (Saint Louis, Mo.), R&Dsystems (Minneapolis, Minn.), Pharmacia Biotech (Piscataway, N.J.),CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Aldrich ChemicalCompany (Milwaukee, Wis.), GIBCO BRL Life Technologies, Inc.(Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.),as well as many other commercial sources known to one of skill.

The nucleic acids (e.g., promoters and vectors) used in the presentmethod can be isolated from natural sources, obtained from such sourcesas ATCC or GenBank libraries, or prepared by synthetic methods.Synthetic nucleic acids can be prepared by a variety of solution orsolid phase methods. Detailed descriptions of the procedures for solidphase synthesis of nucleic acids by phosphite-triester, phosphotriester,and H-phosphonate chemistries are widely available. See, for example,Itakura, U.S. Pat. No. 4,401,796; Carruthers, et al., U.S. Pat. Nos.4,458,066 and 4,500,707; Beaucage & Carruthers; Matteucci; Carruthers,et al., Genetic Engineering 4:1 (1982); Jones, chapter 2, Atkinson, etal., chapter 3, and Sproat, et al., chapter 4, in OLIGONUCLEOTIDESYNTHESIS: A PRACTICAL APPROACH, Gait (ed.), IRL Press, Washington D.C.(1984); Froehler, et al., Tetrahedron Lett. 27:469 (1986); Froehler, etal., Nucleic Acids Res. 14:5399 (1986); Sinha, et al., Tetrahedron Lett.24:5843 (1983); and Sinha, et al., Nucl. Acids Res. 12:4539-4557 (1984),which are incorporated herein by reference.

There are several well-known methods of introducing nucleic acids intobacterial cells, any of which may be used in the present invention (seeSambrook, et al.). These can include fusion of the recipient cells withbacterial protoplasts containing the DNA, DEAE dextran, infection withviral vectors, and the like.

The in vitro delivery of nucleic acids into bacterial hosts can be toany cell grown in culture. Contact between the cells and the geneticallyengineered nucleic acid constructs, when carried out in vitro, takesplace in a biologically compatible medium. The concentration of nucleicacid varies widely depending on the particular application, but isgenerally between about 1 μM and about 10 mM. Treatment of the cellswith the nucleic acid is generally carried out at physiologicaltemperatures (about 37° C.) for periods of time of from about 1 to 48hours, preferably of from about 2 to 4 hours.

Bacterial strains which can be used to express exogenous nucleic acidinclude Escherichia coli, Streptococcus cremoris, Streptococcus lactis,Streptococcus thermophilus, Leuconostc citrovorum, Leuconostocmesenteroides, Lactobacillus acidophilus, Lactobacillus lactis,Bifidobacterium bifidum, Bifidobacteriu breve, and Bifidobacteriumlongum.

In addition to bacterial expression systems, the THP of this inventioncan be expressed in other systems, in particular yeast and baculovirus,but also in mammalian and plant cells. The system used will depend onthe lack of success in other systems, the ability to fold the THPproperly, and the eventual use of the THP. For example, if the THP areto be used to protect bread dough yeast from freezing (see, U.S. PatentNo. 5,118,792), a yeast system will be used. Yeast strains which can beused to express exogenous nucleic acid include Torulopsis holmil,Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyces lactis,and Candida pseudotropicalis. If plants which can live through freezingtemperatures are desired, transgenic techniques can be used to maketransgenic plants. In addition, as will be described below, there may beuses for THP in animals, including insects, fish and crustaceans.However, of course, the system used should give THP with comparablethermal hysteresis activity to that found in the Tenebrio larvae.

As an example of alternative expression systems, InternationalPublication WO 96/11586 (U.S. patent application Ser. No. 08/321,991,filed Oct. 12, 1994), which is incorporated by reference herein,describes the use of fish AFP-transformed Lactobacillus bulgaricus andStreptococcus thermophilus to secrete AFP in order to prevent icerecrystallization in fermented frozen foods, in particular frozenyogurt.

1. Preparation of Recombinant Vectors

To use isolated sequences in the above techniques, recombinant DNAvectors suitable for transformation of cells are prepared. Techniquesfor transforming a wide variety of animal and plant cells are well knownand described in the technical and scientific literature. See, forexample, Weising, et al., Ann. Rev. Genet. 22:421 (1988) for plant cellsand Sambrook for animal and bacterial cells.

A DNA sequence coding for the desired antifreeze protein, for example acDNA sequence encoding the full length THP, will preferably be combinedwith transcriptional and translational initiation regulatory sequenceswhich will direct the transcription of the sequence from the gene in thecells or the intended tissues of the transgenic higher organism. A widevariety of well known transcriptional regulatory elements such aspromoters and enhancers can also be included in the vectors selected toexpress a THP of the invention. Promoters which direct the THP of thisinvention in their native state can be identified by analyzing the 5′sequences of a genomic clone corresponding to the antifreeze proteingenes described herein. Sequences characteristic of promoter sequencescan be used to identify the promoter. Sequences controlling eukaryoticgene expression have been extensively studied. For instance, promotersequence elements include the TATA box consensus sequence (TATAAT),which is usually 20 to 30 base pairs upstream of the transcription startsite. In most instances the TATA box is required for accuratetranscription initiation.

In construction of recombinant expression cassettes of the invention, apromoter fragment, either related to the THP of this invention orheterologous to the THP, may be employed which will direct expression ofthe gene in all tissues of a transgenic organism. Such promoters arereferred to herein as “constitutive” promoters and are active under mostenvironmental conditions and states of development or celldifferentiation. Examples of constitutive promoters of plants includethe cauliflower mosaic virus (CaMV) 35S transcription initiation region,the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens,and other transcription initiation regions from various plant genesknown to those of skill.

Alternatively, the promoter may direct expression of the polynucleotideof the invention in a specific tissue (tissue-specific promoters) or maybe otherwise under more precise environmental control (induciblepromoters). Examples of tissue-specific plant promoters underdevelopmental control include promoters that initiate transcription onlyin certain tissues, such as fruit, seeds, or flowers. The tissuespecific E8 promoter from tomato is particularly useful for directinggene expression so that a desired gene product is located in fruits.Other suitable promoters include those from genes encoding embryonicstorage proteins. Examples of environmental conditions that may affecttranscription by inducible promoters include anaerobic conditions,elevated temperature, or the presence of light.

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes of the invention will typically comprise a marker gene whichconfers a selectable phenotype on transformed cells. For example, themarker may encode antibiotic resistance, particularly resistance tokanamycin. G418, bleomycin or hygromycin.

Plants can be transformed using viral vectors, such as, for example, thetobacco mosaic virus, to express THP proteins of the invention.Selection and construction of vectors and techniques for transforming awide variety of plant cells are well known, for example, see Hamamoto,et al., U.S. Pat. No. 5,618,699.

2. Production of Transgenic Organisms

DNA constructs of the invention may be introduced into the genome of ahost by a variety of conventional techniques. For example, in plants,the DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation and microinjectionof plant cell protoplasts, or the DNA constructs can be introduceddirectly to plant tissue using ballistic methods, such as DNA particlebombardment. As discussed above, plant virus vectors such as tobaccomosaic virus containing the THP sequences of the invention can be usedto innoculate a plant. Alternatively, the DNA constructs may be combinedwith suitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria.

Agrobacterium tumefaciens-mediated transformation techniques, includingdisarming and use of binary vectors, are well described in thescientific literature. See, for example Horsch, et al., Science 233:496(1984), and Fraley, et al., Proc. Nat'l. Acad. Sci. USA 80:4803 (1983).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype,such as increased tolerance to freezing. The transformed plants of theinvention can also be employed as “living factories” to express anantifreeze protein in substantial quantities. Such plant regenerationtechniques rely on manipulation of certain phytohormones in a tissueculture growth medium, typically relying on a biocide and/or herbicidemarker which has been introduced together with the desired nucleotidesequences. Plant regeneration from cultured protoplasts is described inEvans, et al., PROTOPLASTS ISOLATION AND CULTURE, HANDBOOK OF PLANT CELLCULTURE, pp. 124-176, Macmillian Publishing Company, New York, 1983; andBinding, REGENERATION OF PLANTS, PLANT PROTOPLASTS, pp.21-73. CRC Press,Boca Raton, 1985. Regeneration can also be obtained from plant callus,explants, organs, or parts thereof. Such regeneration techniques aredescribed generally in Klee, et al., Ann. Rev. of Plant Phys. 38:467(1987).

To produce a transgenic plant or animal, for example a salt-water fish,microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsinto cells using polyethylene glycol precipitation is described inPaszkowski, et al., EMBO J. 3:2717 (1984). Electroporation techniquesare described in Fromm, et al., Proc. Natl. Acad. Sci. USA 82:5824(1985). Ballistic transformation techniques are described in Klein, etal., Nature 327:70 (1987).

III. DETECTION AND CHARACTERIZATION OF THE CLASS OF THP OF THISINVENTION

By the assays described below, the THP of this invention sharecharacteristics with a thermal hysteresis protein isolated from finallarval instar Tenebrio molitor hemolymph. These assays are used todefine whether other novel THP are sufficiently related to the prototypeproteins YL-1 through YL-4 so as to fall within the scope of thisinvention. The assays can also be used to detect and quantify THPspresent in bacteria broth, tissue culture fluid and plant and animaltissues.

A. Detection of THP

Expressed THP may be detected or quantified by a variety of methods.Preferred methods involve the use of functional activity assays andimmunological assays utilizing specific antibodies.

1. Antibodies

Methods of producing polyclonal and monoclonal antibodies are known tothose of skill in the art. See, e.g., Coligan, CURRENT PROTOCOLS INIMMUNOLOGY, Wiley/Greene, NY (1991); Stites et al. (eds.) BASIC ANDCLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein (“Stites”); Goding, MONOCLONALANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York,N.Y. (1986); Kohler & Milstein, Nature 256:495 (1975); and Harlow andLane. Such techniques include antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors. See, Huse et al., Science 246:1275 (1989) (“Huse”); and Ward etal., Nature 341:544 (1989).

To produce large amounts of antibodies for use in, for example,immunoaffinity purification, a number of immunogens may be used. THPfrom Tenebrio molitor or from the transformed cells as described in thisinvention are the preferred immunogens for the production of monoclonalor polyclonal antibodies. Naturally occurring antifreeze protein fromother organisms may also be used either in pure or impure form.Synthetic peptides made using a fragment of antifreeze protein sequencedescribed herein may also used as an immunogen for the production ofantibodies to the protein. The peptides can be used alone or conjugatedto another composition.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen is mixed with an adjuvant, asdescribed above, and animals are immunized. The animal's immune responseto the immunogen preparation is monitored by taking test bleeds anddetermining the titer of reactivity to the immunogen. When appropriatelyhigh titers of antibody to the immunogen are obtained, blood iscollected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired. (See Harlow and Lane, supra).

Large amounts of monoclonal antibodies for use in immunoaffinitypurification or immunoassays may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antifreeze protein are immortalized,commonly by fusion with a myeloma cell (See, Kohler & Milstein, Eur. J.Immunol. 6:511 (1976), incorporated herein by reference). Alternativemethods of immortalization include transformation with Epstein BarrVirus, oncogenes, or retroviruses, or other methods well known in theart. Colonies arising from single immortalized cells are screened forproduction of antibodies of the desired specificity and affinity forTHP. The yield of the monoclonal antibodies produced by such cells maybe enhanced by various techniques, including injection into theperitoneal cavity of a vertebrate host. Alternatively, one may isolateDNA sequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according to thegeneral protocol outlined in Huse.

The concentration of THP can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Stites. Moreover, the immunoassays of the present inventioncan be performed in any of several configurations, which are reviewedextensively in ENZYME IMMUNOASSAY, E. T. Maggio, ed., CRC Press, BocaRaton, Fla. (1 980); Tijssen; and Harlow and Lane, each of which isincorporated herein by reference.

For example, in order to produce antisera for use in an immunoassay forantifreeze protein, YL-1, YL-2, YL-3, YL-4 or fragments thereof, areisolated as described herein. An inbred strain of mice or rabbits isimmunized with the above antifreeze isoforms or polypeptide of SEQ IDNO:4 using a standard adjuvant, such as Freund's adjuvant, and astandard immunization protocol. Alternatively, a synthetic peptidederived from the sequences disclosed herein and conjugated to a carrierprotein can be used an immunogen. Polyclonal sera are collected andtitered against the THP in an immunoassay, for example, a solid phaseimmunoassay with the THP immobilized on a solid support. Polyclonalantisera with a K_(a) of 10⁻¹ M⁻¹ or greater are selected and tested fortheir cross reactivity against homologous proteins from other organismsand/or non-antifreeze protein, using a competitive binding immunoassay.Specific monoclonal and polyclonal antibodies and antisera will usuallybind with a K_(D) of at least about 0.1 mM, more usually at least about1 μM, preferably at least about 0.1 μM or better, and most preferably,0.01 μM or better.

2. Immunological Binding Assays

In a preferred embodiment, THP are detected and/or quantified using anyof a number of well recognized immunological binding assays (see, e.g.,U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For areview of the general immunoassays, see also METHODS IN CELL BIOLOGYVol. 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. NewYork (1993); and Stites. Immunological binding assays (or immunoassays)typically utilize a “capture agent” to specifically bind to and oftenimmobilize the analyte (in this case THP or a fragment thereof). Thecapture agent is a moiety that specifically binds to the analyte. In apreferred embodiment, the capture agent is an antibody that specificallybinds to THP. The antibody (anti-THP) may be produced by any of a numberof means well known to those of skill in the art and as described above.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledTHP polypeptide or a labeled anti-THP antibody. Alternatively, thelabeling agent may be a third moiety, such as another antibody, thatspecifically binds to the antibody/THP complex.

In a preferred embodiment, the labeling agent is a second THP antibodybearing a label. Alternatively, the second THP antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al., J. Immunol. 111:1401-1406 (1973), andAkerstrom, et al., J. Immunol. 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing, steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 4° C. to 40° C.

a. Non-Competitive Assay Formats.

Immunoassays for detecting THP may be either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte (in this case THP) is directly measured. Inone preferred “sandwich” assay, for example, the capture agent (anti-THPantibodies) can be bound directly to a solid substrate where they areimmobilized. These immobilized antibodies then capture protein presentin the test sample. The THP thus immobilized is then bound by a labelingagent, such as a second THP antibody bearing a label. Alternatively, thesecond THP antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second can be modified with adetectable moiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

b. Competitive Assay Formats.

In competitive assays, the amount of analyte (THP) present in the sampleis measured indirectly by measuring the amount of an added (exogenous)analyte (THP) displaced (or competed away) from a capture agent (antiTHP antibody) by the analyte present in the sample. In one competitiveassay, a known amount of, in this case THP, is added to the sample andthe sample is then contacted with a capture agent, in this case anantibody that specifically binds THP. The amount of THP bound to theantibody is inversely proportional to the concentration of THP presentin the sample.

In a particularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of THP bound to the antibody may bedetermined either by measuring the amount of THP present in anTHP/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed THP. The amount of THP may be detected byproviding a labeled THP molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay a known analyte, in this case THP, is immobilized on a solidsubstrate. A known amount of anti-THP antibody is added to the sample,and the sample is then contacted with the immobilized THP. In this case,the amount of anti-THP antibody bound to the immobilized THP isinversely proportional to the amount of THP present in the sample. Againthe amount of immobilized antibody may be detected by detecting eitherthe immobilized fraction of antibody or the fraction of the antibodythat remains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Immunoassays in the competitive binding formant can be used forcrossreactivity determinations to permit one of skill to determine if anovel THP is sufficiently related to the claimed THP so as to fall underthe claims of this invention. For example, a THP fragment of SEQ ID NO:4can be immobilized to a solid support. Proteins are added to the assaywhich compete with the binding of the antisera to the immobilizedantigen. The ability of the proteins to compete with the binding of theantisera to the immobilized THP is compared to the binding by the sameTHP as was used to coat the solid support. The percent crossreactivityfor the above proteins is calculated, using standard calculations. Thoseantisera with less than 10% crossreactivity with the THP of SEQ ID NO:4are selected and pooled. The cross-reacting antibodies are optionallyremoved from the pooled antisera by immunoabsorption with the aboveproteins.

The immunoabsorbed and pooled antisera can be used in a competitivebinding immunoassay, as described above, to analyze whether a secondprotein is an antifreeze protein of this invention. In the competitivebinding immunoassay the protein, or immunogen, used to develop theantiserum competes with a second, uncharacterized protein or peptide inan antibody binding reaction. The two proteins are each assayed at awide range of concentrations and the amount of each protein required toinhibit 50% of the binding of the antisera to the immobilized protein isdetermined. If the amount of the second protein required is less than 10times the amount of the characterized immunogen (for example, SEQ IDNO:2, SEQ ID NO:4 or an immunogenic fragment thereof) that is required,then the second protein is said to specifically bind to an antibodygenerated to that characterized (antifreeze protein) immunogen.

c. Other Assay Formats.

Western blot (immunoblot) analysis can be used to detect and quantifythe presence of antifreeze protein in a sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind THP. The anti-THP antibodies specifically bind toTHP on the solid support. These antibodies may be directly labeled oralternatively may be subsequently detected using labeled antibodies(e.g., labeled sheep anti-mouse antibodies) that specifically bind totile anti-antifreeze protein.

In addition to using nucleic acid probes for identifying novel forms ofthe class of proteins claimed herein, it is possible to use antibodiesto probe expression libraries. This is a well known technology (SeeYoung & Davis, Proc. Nat'l Acad. Sci. USA 80:1194 (1982)). In general, acDNA expression library may be prepared from commercially available kitsor using readily available components. Phage vectors are preferred, buta variety of other vectors are available for the expression of protein.Such vectors include but are not limited to yeast, animal cells andXenopus oocytes. One selects mRNA from a source that is enriched withthe target protein and creates cDNA which is then ligated into a vectorand transformed into the library host cells for immunoscreening.Screening involves binding and visualization of antibodies bound tospecific proteins on cells or immobilized on a solid support such asnitrocellulose or nylon membranes. Positive clones are selected forpurification to homogeneity and the isolated cDNA then prepared forexpression in the desired host cells. A good general review of thistechnology can be found in METHODS OF CELL BIOLOGY, VOL. 37 entitledAntibodies in Cell Biology, Assai (ed.) 1993.

Where the antibodies are generated to protein like the THPs of thisinvention, which are rich in cysteine and hypothetically contain manydisulfide bridges, the test proteins are optionally denatured to fullytest for selective binding and it may be best to measure the testproteins against proteins of similar size, e.g., one would test a fulllength THP against a prototype full length THP even though the antiserawas generated against a fragment of the prototype THP. This simplifiesthe test and avoids having to take into account conformational problemsand molecular weight/molar concentrations in the determination of theresults from the competitive immunoassays.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,Amer. Clin. Prod. Rev. 5:34 (1986)).

B. Purification of THP

The polypeptides of this invention may be purified to substantial purityby standard techniques, from a variety of sources such as larvalhemolymph, tissue culture media, transgenic plants and animals, yeastsand bacteria. For standard purification procedures, including selectiveprecipitation with such substances as ammonium sulfate; columnchromatography, immunopurification methods, and others see, forinstance, Scopes, PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE,Springer-Verlag: New York (1982), U.S. Pat. No. 4,673,641, Ausubel, andSambrook, all incorporated herein by reference. 1. Purification of THPfrom Bacterial Cultures

In the case of secreted proteins, the protein of interest can beisolated and purified from the broth in which bacteria have been grownwithout having to resort to the cell lysis methods detailed below.

2. Purification of THP from Bacterial Cytoplasm and Periplasm

After expression of THP in E. coli, the protein may be found in theperiplasm, cytoplasm or inclusion bodies of the bacteria. Theperiplasmic fraction of the bacteria can be isolated by cold osmoticshock in addition to other methods known to those of skill in the art(see Ausubel, and Trayer, & Buckley, J. Biol. Chem. 245(18):4842(1970)).

To isolate proteins from the periplasm and cytoplasm, the bacterialcells are centrifuged to form a pellet. The pellet is resuspended in abuffer containing 20% sucrose. To lyse the cells, the bacteria arecentrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ andkept in an ice bath for approximately 10 minutes. The cell suspension iscentrifuged and the supernatant decanted and saved. Alternatively, thebacteria can be lysed by sonication. The proteins present in thesupernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

3. Purification of Inclusion Bodies

When recombinant proteins are expressed by the transformed bacteria inlarge amounts, typically after promoter induction; but expression can beconstitutive, the proteins may form insoluble aggregates.

Purification of aggregate proteins (hereinafter referred to as inclusionbodies) involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, typically but notlimited by, incubation in a buffer of about 100-150 μg/mL lysozyme and0.1% NONIDET P40®, a non-ionic detergent. The cell suspension can beground using a Polytron grinder (Brinkman Instruments, Westbury, N.Y.).Alternatively, the cells can be sonicated on ice. Alternate methods oflysing bacteria are described in Ausubel and Sambrook and will beapparent to those of skill in the art.

The cell suspension is centrifuged and the pellet containing theinclusion bodies resuspended in buffer, e.g., 20 mM Tris-HCl (pH 7.2), 1mM EDTA, 150 mM NaCl and 2% TRITON-X 100®, a non-ionic detergent. It maybe necessary to repeat the wash step to remove as much cellular debrisas possible. The remaining pellet of inclusion bodies may be resuspendedin an appropriate buffer (e.g. 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties) together with a reducing agent such as DTT. Theproteins that formed the inclusion bodies can then be renatured bydilution or dialysis with a compatible buffer. Suitable solventsinclude, but are not limited to urea (from about 4 M to about 8 M),formamide (at least about 80%, volume/volume basis), and guanidinehydrochloride (from about 4 M to about 8 M). Some solvents which arecapable of solubilizing aggregate-forming proteins, for example SDS(sodium dodecyl sulfate), 70% formic acid, are inappropriate for use inthis procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of immunologically and/or biologically activeprotein.

After solubilization, the protein can be separated from other bacterialproteins by standard separation techniques.

4. Standard Protein Separation Techniques

a. Solubility Fractionation

Often as an initial step and if the protein mixture is complex, aninitial salt or organic solvent fractionation can separate many of theunwanted host cell proteins (or proteins derived from the cell culturemedia) from the recombinant protein of interest. The preferred salt isammonium sulfate. Ammonium sulfate precipitates proteins by effectivelyreducing the amount of water in the protein mixture. Proteins thenprecipitate on the basis of their solubility. The more hydrophobic aprotein is, the more likely it is to precipitate at lower ammoniumsulfate concentrations. A typical protocol is to add saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This will precipitate the mosthydrophobic of proteins. The precipitate is discarded (unless theprotein of interest is hydrophobic) and ammonium sulfate is added to thesupernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

b. Size Differential Filtration

If the size of the protein of interest is known or can be estimated fromthe cDNA sequence, proteins of greater and lesser size can be removed byultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

c. Column Chromatography

Proteins can be separated on the basis of their size, net surfacecharge, hydrophobicity and affinity for ligands. In addition, antibodiesraised against proteins can be conjugated to column matrices and theproteins immunopurified. All of these methods are well known in the art.See Scopes, R. K., Protein Purification: Principles and Practice, 2nded., Springer Verlag, (1987).

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

In a preferred embodiment, the purification of THP from E. colisupernatant is accomplished in part by gel filtration, and proteinconcentrations are determined according to a number of techniques. e.g.,Bradford, Anal. Biochem. 72:248-257 (1976).

d. Amino Acid Sequence

The amino acid sequences of the THP of this invention can be determinedby, for example, Edman degradation, a technique which is well known inthe art. By Edman degradation, an internal section of THP was sequencedspanning a repeating motif of 12 contiguous amino acids which was foundto make up the bulk of the THP. This motif (SEQ ID NO:1) is repeated atleast 5 times and more preferably 5 to 12 times in each isoform.

In addition to the internal sequencing, N-terminal sequencing can beperformed by techniques known in the art. However, in the native THP26and by analogy in 3 of the recombinant THP isoforms, the N terminus wasblocked. By nucleic acid sequencing, the N termini were determined to bea glutamine which would be consistent with N-terminal sequencing beingblocked by pyroglutamate (cyclized glutamine). The deduced N-termini ofthe class of THP of this invention is identified as SEQ ID NO:4.

e. Molecular Weight/Isoelectric Point

The molecular weight of a protein can be determined by many differentmethods, all known to one of skill in the art. Some methods ofdetermination include: SDS gel electrophoresis, native gelelectrophoresis, molecular exclusion chromatography, zonalcentrifugation, mass spectroscopy, and calculation from sequencing.Disparity between results of different techniques could be due tofactors inherent in the technique. For example, native gelelectrophoresis, molecular exclusion chromatography, zonalcentrifugation depend factors such as the size, shape and net charge ofthe protein. The proteins of this invention are rich in cysteine andmight be expected to form many disulfide bonds, both intra- andintermolecular. SDS gel electrophoresis depends on the binding of SDS toamino acids present in the protein. Some amino acids bind SDS moretightly than others, therefore, proteins will migrate differentlydepending on their amino acid composition. In the instant invention,because the THP disclosed herein are repeating units of a motif, therelative number of different amino acids is quite small and could resultin a large disparity in molecular weight by SDS gel electrophoresis. Thecalculated molecular weight from the sequence utilizes the frequency ofamino acids present in the protein and multiplies that frequency by themolecular weight of the amino acid. If the protein is glycosylated,calculated molecular weight will not reflect this and mass spectroscopymay be a necessary alternative.

In the present invention, the apparent molecular weight of the class ofTHP by molecular exclusion chromatography was between 17 and 19 kD; bySDS gel electrophoresis, 22-30 kD. By mass spectroscopy, the molecularweight of the class of proteins was found to be between 7 to 12,preferably 8 to 11 and most preferably from about 8.4 to 11.7 kD. Thisagreed with the results obtained when the molecular weight wascalculated from the amino acid sequencing. By comparing the amino acidsequences and the molecular weights by mass spectroscopy, it is likelythat the differences in molecular weight are due largely to differentnumber of motif repeats.

The isoelectric point of a protein can be determined by native gel (ordisc) electrophoresis, isoelectric focussing or in a preferred method,by calculation given the amino acid content of the protein. The class ofproteins of the instant invention have a calculated pI of about 8 to 10.

f. Functional Assays

The THP species and isoforms of the invention can be identified andcharacterized by at least two functional properties, for example,thermal hysteresis and unique formation of ice crystals:

(1) Thermal Hysteresis

The proteins of this invention are approximately 100 fold more activethan known fish antifreeze proteins in a thermal hysteresis assay.Thermal hysteresis is defined as the difference between the solutionfreezing and melting temperatures. Freezing point is taken as thetemperature at which uncontrollable ice growth occurs from a seed icecrystal. Melting point is taken as the warmest temperature at which anice crystal can be stably held without melting. TH activity can bemeasured in a nanoliter osmometer (Clifton Technical Physics, Martford,N.Y.) by methods well known in the art (see, for example. Chakrabartty &Hew, Eur. J. Biochem. 202:1057 (1991)). The starting ice crystal isusually 20-50 μm in diameter. The buffer used is typically 100 mMNH₄HCO₃ (pH 7.9) but other buffers of similar osmolarity can be used.Alternatively, TH activity can be measured in bacterial broth, tissueculture fluid, or hemolymph.

The osmometer is a thermal electric cooling module with a separate butlinked variable temperature control. This apparatus allow temperatureregulation in the 0° C. to −9° C. range with a deep freeze mode to −40°C. The cooling module can be set up on a microscope stage where thegrowth and melt behavior of an ice crystal can be observed directly. Thesample holder can be a small plate with dimensions of about 7 mm×7mm×0.75 mm containing multiple small sample holes (about 0.35 mm indiameter). A drop of immersion oil, such as Cargille's B immersion oil,can be placed on the underside of the sample holder so that the sampleholes are filled. 1 to 5 nL samples are then delivered into the centerof the oil-filled hole by a capillary tube.

To measure TH activity, the samples are first frozen by cooling thesamples rapidly to −40° C. and then allowing them to warm up to themelting point temperature. Once the melting point is reached, thesamples are cooled by approximately 0.02° C. (10 milli-osmoles ormosmoles) per 10-15 seconds until the freezing temperature is reached.The conversion from the unit “osmos” to “° C.” is 1.00 osmoles equals1.86° C. In most instances, when the freezing point of a THP sample isreached, the ice crystal within the sample will grow spontaneously andrapidly. This leads to freezing of the entire sample.

Alternatively, a small ice crystal can be frozen onto the surface of asolution and the temperature of the solution immediately reduced tobelow freezing. The temperature below freezing when the nucleated icecrystal begins to grow is the freezing point depression or the thermalhysteresis measurement (see, Patterson & Duman, J. Exp. Zool. 210:361(1979); and Wu, et al., J. Comp. Physiol. B 161:271 (1991). The thermalhysteresis activity of a solution is dependent on the concentration ofthe antifreeze protein, with the greater the concentration of theprotein, the greater the activity shown by the solution. However,increased concentrations of THPs produce incrementally smaller increasesin TH activity and a maximum is approached. In other words, therelationship between THP concentration and TH is hyperbolic, not linear.

The proteins of the present invention preferably have thermal hysteresisvalues greater than 1.0° C. at about 1 mg/mL, more preferably greaterthan 1.5° C. at about 1 mg/mL and most preferably between about 1.5-3.0°C. at about 1 mg/mL.

(2) Unique Formation of Ice Crystals

In addition to having approximately 100 fold more specific activity thanpreviously known fish antifreeze proteins, the proteins of thisinvention produce different shaped ice crystals. Under microscopicanalysis, fish antifreeze proteins produce ice crystals which arehexagonal bipyramids with flat, well-defined facets. The THP of thisinvention, on the other hand, form crystals with rounded edges,preferably oval-shaped and with non-flat surfaces, preferably convex.

IV. USE OF ANTIFREEZE PROTEINS AND RELATED GENES

The proteins or genes encoding the THP may be used in ways to suppressice crystal growth. For a comprehensive review of uses of antifreezeproteins, see U.S. Pat. No. 5,118,792. The THP of this invention may beintroduced in the protein form, or they may be introduced as genes whichare expressed endogenously at a level which should be attainable byexpressing an antifreeze protein in a cell under the control of asuitable strong promoter to produce the proteins. Suitableconcentrations of THP will vary depending on the use, but will typicallybe in the range of from about one part per billion to about one part perthousand (i.e. 1 μg/L to 1 g/L).

In one embodiment of the invention, the proteins will be introduced tofoodstuffs. This has a number of different aspects. One is theintroduction into plant foodstuffs, either into the entire plant andthus conferring some degree of general resistance to damage fromsubfreezing climatic conditions, or into a plant part such as the fruitor vegetable portion to minimize damage specifically to those particularplant organs upon freezing. Exemplary plant parts are stems, roots,leaves, flowers, petioles, pericarp, seeds, vegetative tissue, tubersand so forth.

The texture, taste, and useful storage life of frozen vegetables will beimproved, for example, celery, potatoes, asparagus, peas, carrots,beans, broccoli, sweet corn and spinach. Similarly, the texture, tasteand useful storage life of fruits will be enhanced, includingstrawberries, blueberries, raspberries, citrus fruits, bananas, grapes,kiwis, peaches, pineapples, plums, cherries, tomatoes and mangoes.

This introduction into plant and other products may be most easilyaccomplished by genetic introduction of appropriate nucleic acids intothe target organism. Expression of the nucleic acid, eitherconstitutively or inducibly, before food processing has begun, or afterharvesting and processing has begun, may lead to sufficiently highlevels of the polypeptide to effectively protect the foodstuff, such asup to 0.5%, but more preferably up to about 0.1% of total plant proteinby mass. Expression can also be on a tissue specific basis. For example,linkage to ripening genes in fruits may result in expression even afterharvesting from the producing plant.

The polypeptides may also be added into foods which are expected to befrozen. Many frozen foods are intended to be consumed in the cold state,for example, ice cream, frozen yogurt, ice milk, sherbet, popsicles,frozen whipped cream, frozen cream pies, frozen puddings and the like.In particular, texture and flavor are adversely affected by theformation of large ice crystals throughout a freeze-thaw cycle thatoccurs in most home frost-free freezers or upon sustained storage in thefrozen state. This ice crystal growth process may be prevented entirely,or at least minimized by tile addition of antifreeze polypeptides. Thepurified antifreeze protein may be either incorporated throughout thefoodstuff, or may, alternatively, be applied to the surface wherecondensation and crystal formation is expected to occur most readily.

In another embodiment, the genes that encode the THP of this inventionare used to transform microorganisms which when added to foodstuffs,protect the foodstuffs or the microorganism from freezing. For example,bacteria such as Streptococcus thermophilus and Lactobacillus bulgaricuscan be added to dairy products that is intended to be sold as frozenyogurt. In addition to fermenting the dairy products to produce yogurt,the THP expressed by the bacteria will protect the product from homefreezer freeze-thaw cycles and produce a more palatable product.

Another use would be to transform dough yeast with nucleic acidsencoding THP. Upon incorporation and expression of this gene into theyeast, and use of these yeast in frozen dough, the dough will naturallyleaven upon thawing because the yeast viability will remain high uponthawing. Because less damage accumulates from storage in the presence ofthese antifreeze polypeptides and thawed samples preserve highviability, either longer storage times will be possible, or perhaps muchsmaller aliquots will need to be stored.

There are various embodiments not specific to the food freezer. One isthe use of THP to protect plants from climatic freezing conditions. TheTHP may be either internally incorporated into the cytoplasm byexpression of an introduced gene, or the proteins may be externallyapplied to the plants. External application may be achieved either bydirect application of the proteins to the plant, or by the externaldeposit onto the plant of an organism which secretes the proteins. Thesesame alternatives for introduction apply to other uses as well.

In addition to plants, it is envisioned that the THP of this inventioncan be used to produce transgenic animals, including fish that canwithstand sub-zero temperatures. While some polar fish do synthesizeantifreeze proteins, most fish do not live in environments where thewater temperature drops below 0° C. However, transgenic fish containingexogenous nucleic acid encoding THP could be held and perhaps partiallyraised in sub-zero salt-water. In particular, salt-water fish beingraised in farms, most particularly salmon. In addition to salmon, farmedtransgenic prawns are a potential recipient of THP genes.

In addition to the above embodiments, it is envisioned that the THP ofthis invention can be used to regulate the expression of endogenous THPgenes within a cold-tolerant organism. The expression of antifreezeprotein gene products may be increased as a method of preparing, e.g.,for subsequent isolation, endogenous antifreeze proteins. Conversely, bydownregulating endogenous antifreeze gene expression, an otherwisecold-tolerant pest may be converted to a less aggravating phenotype.

Methods of altering the expression of endogenous genes are well known tothose of skill in the art. Typically such methods involve altering orreplacing all or a portion of the regulatory sequences controllingexpression of the particular gene that is to be regulated. In apreferred embodiment, the regulatory sequences (e.g., the nativepromoter) upstream of one or more of the THP are altered.

This is typically accomplished by the use of homologous recombination tointroduce a heterologous nucleic acid into the native regulatorysequences. To downregulate expression of one or more THP gene products,simple mutations that either alter the reading frame or disrupt thepromoter are suitable. To upregulate expression of the THP geneproducts, the native promoter(s) can be substituted with heterologouspromoter(s) that induce higher than normal levels of transcription.

In a particularly preferred embodiment, nucleic acid sequencescomprising the structural gene in question or upstream sequences areutilized for targeting heterologous recombination constructs. Utilizingthe structural gene sequence information provided in SEQ ID NOs:2 and 5or the upstream or downstream sequence information provided in SEQ IDNos:10, 12, 13 and 14, one of skill in the art can create homologousrecombination constructs with only routine experimentation.

The use of homologous recombination to alter expression of endogenousgenes is described in detail in U.S. Pat. No. 5,272,071, WO 91/09955, WO93/09222, WO 96/29411, WO 95/31560, and WO 91/12650. Homologousrecombination in mycobacteria is described by Azad, et al., Proc. Nat'lAcad. Sci. USA 93:4787 (1996); Baulard, et al., J. Bacteriol. 178:3091(1996); and Pelicic, et al. Mol. Microbiol. 20:919 (1996). Homologousrecombination in animals has been described by Moynahan, et al., in Hum.Mol. Genet. 5(7):875 (1996) and in plants by Offringa, et al. EMBO J.9(10):3077 (1990).

Another embodiment is the introduction of an antifreeze protein intoaqueous liquids surrounding an organ, tissue or other biological sample.One particular use would be during transportation to a hospital for atransplantation operation or for storage purposes. The antifreezeprotein should allow short- or long-term storage at a subfreezingtemperature, thereby minimizing inherent metabolism or degradation, butwith substantially diminished cellular damage from ice crystal growth.Other medically important temperature sensitive biological samples areblood and blood products, therapeutic agents, protein drugs, bioassayreagents and vaccines.

Yet another embodiment is the introduction of an antifreeze protein intocells or their extracts destined for frozen storage. For example,bacterial cells, yeast cells, plant cells and, most particularly, animalcells containing the THP have increased cell or tissue viability withminimal or no loss of inherent characteristics due to the freeze-thawprocess. Subcellular samples or cellular extracts may have similarsensitivities to freezing, especially on prolonged storage. Typicalexamples will be in vitro protein translation systems, enzymepreparations, and particularly samples which contain sensitive membranecomponents, such as chloroplast or mitochondrial membrane preparations.In particular, samples containing organelles may display increasedresistance to freezing damage upon addition of these antifreezepolypeptides. Soft animal tissues will exhibit less damage upon freezingin the presence of the subject polypeptides, and addition of thepolypeptides will be useful in situations when cellular integrity uponfreezing and subsequent thawing is important or desired, such as fortissue culture deposits. Thus, samples destined for frozen storage, suchas for cell or tissue depositories, might routinely have the proteinsadded to them. Among the cell types often stored are genetic variants ofbacteria, fungi (including yeast), and, particularly, higher eukaryotecells (such as hybridoma strains and tissue culture cell lines).

Also included in the invention are compositions and uses based on themixture of THP with stabilizers well known to those skilled in the artand other additives. These compounds may be present to inhibit decay,inhibit oxidation, prevent discoloration, inhibit microbial growth,stabilize emulsions and so forth.

Also included in the invention are compositions based on THPs suitablefor depressing the freezing point or inhibiting freezing in non-organicsystems, such as for use in deicing treatments.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 THP from Tenebrio molitor Larvae

The following example details the isolation of THP from the hemolymph ofTenebrio molitor. Tenebrio molitor larvae were reared as previouslydescribed (see, Graham, et al., Insect Biochem. Molec. Biol. 26:127(1996)) but in darkness. Hemolymph (600 μL) was obtained from exudatesat severed prolegs of 100 presumed final instar larvae and diluted 1:1in ice-cold hemolymph buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 1mM phenylthiocarbamide.

Diluted hemolymph (1.2 mL) was loaded onto an S-100 SEPHACRYL®(Pharmacia) column (92 cm×1.6 cm) and eluted with hemolymph bufferwithout phenylthiocarbamide. Selected active fractions were combined andchromatographed by reversed-phase HPLC on a C18 analytical column(Vidac), using a gradient of 0.4% acetonitrile/min in 0.05%trifluoroacetic acid. Fractions were lyophilized and resuspended in 50μL of 0.1 M NH₄HCO₃ (pH 8.0) for TH measurements following standardprocedures (see, Chakrabarrty & Hew, Eur. J. Biochem. 202:1057 (1991)).Ice crystal morphology was photographed using black and white film, andcrystal c and a axes were identified using cross-polarized light (Hobbs,ICE PHYSICS, Clarendon Press, London (1974)). Active fractions wereresolved by 15% SDS-PAGE and stained using the Silver Stain Plus Kit(Bio-Rad).

Approximate molecular weights were determined by MALDI massspectrometry. Two active, well-resolved HPLC fractions were subjected toamino acid analysis, N-terminal sequencing and electrospray ionizationmass spectrometry. One fraction was reduced, alkylated, and cut withendoprotease Lys-C. Following separation of the cleavage products byreversed-phase HPLC, the best-resolved fragment was sequenced byautomated Edman degradation.

When diluted larval Tenebrio hemolymph was fractionated by gel exclusionchromatography, there were two overlapping peaks of TH activity withapparent molecular weights of approximately 19 and 17 kD (FIG. 1). Whenthe leading peak was analyzed by HPLC, active proteins eluted between16% and 22% acetonitrile (FIG. 2) and the more abundant but inactiveproteins eluted later in the gradient. Mass spectrometry (FIG. 3)indicated that the inactive proteins (12.2 kDa to 12.4 kDa) migrated onSDS-PAGE according to their molecular mass, while the smaller THproteins (7.3 kDa to 10.7 kDa) migrated anomalously as diffuse bandswith apparent molecular weights ranging from 22 to 30 kDa. Fractionswith the highest TH activity, corresponding to HPLC fractions 26 and 27(FIG. 2), were analyzed for their amino acid content. The compositionsof the two fractions were similar and both were rich in Thr and otheramino acids with short side chains (Gly, Ala, Ser). Overall, theproteins were moderately hydrophilic (Wishard, et al., Comput. Appl.Biosci. 10:121 (1994)). The TH activities of THP26 (fraction 26) at 55μg/mL and of THP27 at 21 μg/mL were 1.6° C. and 1.1° C., respectively,close to the maximum values obtained with 10-30 mg/mL of fish AFPs(Davies & Hew, FASEB J. 4:2460 (1990)).

Both THP26 and THP27 were N-terminally blocked. An internal fragmentfrom THP26 released by endoproteinase Lys-C digestion was sequenced, andits amino acid content, including 7 Thr out of 20 residues, wasconsistent with the overall amino acid composition. Degenerate primersto this sequence were used in conjunction with vector primers togenerate PCR products for screening a Tenebrio cDNA library.

Example 2 cDNA Library Screening for THP-Encoding Genes

To isolate other isoforms of tile THP of this invention,oligonucleotides for probes (SEQ ID NOs:2 and 5), PCR primers (SEQ IDNOs: 6 and 7) and sequencing primers (SEQ ID NOs: 8 and 9) were designedbased on the consensus sequence determined from sequencing TH positiveclones (YL-1-4).

Aliquots of a Tenebrio molitor larval fat body λ-Zap cDNA library (see,Graham, et al., Insect Biochem. Molec. Biol. 26:127 (1996)) werescreened with the nucleic acid sequence of YL-1 from the 5′ end to thestop codon (SEQ ID NO: 16). Approximately 1×10⁵ plaques were screened atmoderate stringency following standard methodologies using the sequencelisted above. Isolated positive plaques were subjected to in vivoexcision using R408 helper phage (Stratagene) as per manufacturer'sinstructions. The double-stranded DNA obtained was purified andsequenced as above using the vector primers T7 and T3 as well as SEQ IDNOs:8 and 9.

The conceptual translations from the nucleic acid sequencing matched thesequenced peptide fragment at up to 18 of the 20 residues. The first 28amino acids represented a secretory signal peptide with Cys at the −3and −1 positions (von Heijne, Nucl. Acids Res. 14:4683 (1986)). TheN-terminal amino acid of three of the variants was predicted to be Gln.This was consistent with N-terminal blockage in which the N-terminal Glnwas converted to pyroglutamate by cyclization. The cleavage site of thefourth variant was not clearly predicted.

Example 3 Subcloning and Protein Expression

PCR linker-primers (SEQ ID NO: 6 and 7) were designed to amplify the THPcoding region and to introduce a Met codon before the presumedN-terminal residue of the mature protein. The resulting fragment wasligated into the pET-20b(+) vector (Novagen) and transformed into E.coli BL21(DE3). Expression was induced using isopropylβ-D-thiogalactopyranoside, cells were harvested by centrifugation, andsonicated in 10 mM Tris-HCl (pH8.0), 1 mM EDTA, 0.1 mMphenylmethylsulfonyl fluoride. Cleared supernatant was loaded onto a G75gel exclusion chromatography column. Active fractions were pooled,lyophilized, resuspended in 50 mM Tris-HCl (pH 8.0), and dialyzedovernight against the same buffer.

Example 4 Functional Activity from Cloned THP

To prove that the cloned sequence codes for a THP, the shortest of thefour cDNAs with a conventional signal cleavage site (YL-2, FIG. 6) wasexpressed in E. coli. TH activity was detected in the supernate of thecell lysate, indicating that some of the protein was able to fold wellenough to display activity. Partially purified recombinant THP showed5.3° C. of TH and was indistinguishable in its properties from the THPin Tenebrio hemolymph. Its activity was eliminated by reduction but wasunaffected by chelation. Also, ice crystals formed in the presence ofdilute hemilolymph (FIG. 4-I) and recombinant protein were identical(FIGS. 4-II). These observations suggest that this THP and its isoformscan account for all the TH activity in the insect. Moreover, extensiveanalysis of hemolymph and total extracts have shown no evidence of THPthat are unrelated to these isoforms.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1-35. (canceled)
 36. An isolated or recombinantly expressed antifreezeprotein, said protein comprising the following: (i) a calculatedmolecular weight of between 7 and 13 kDa; (ii) a thermal hysteresisactivity of greater than 1.5° C. at a concentration of about 1 mg/mL;(iii) the N-terminal amino acid motif set forth in SEQ ID NO:3: (iv) atleast 60% amino acid sequence identity to an antifreeze protein YL-4(SEQ ID NO:15); and (v) at least greater than 1 repeat of the 12contiguous amino acid motif set forth in SEQ ID NO:1.
 37. The isolatedor recombinantly expressed antifreeze protein of claim 36 comprising atleast 5 repeats of the 12 contiguous amino acid motif set forth in SEQID NO:1.
 38. The isolated or recombinantly expressed antifreeze proteinof claim 36 comprising 5 to 12 repeats of the 12 contiguous amino acidmotif set forth in SEQ ID NO:1.
 39. The isolated or recombinantantifreeze protein of claim 36, wherein the calculated molecular weightof the antifreeze protein is between 8 and 12 kDa.
 40. The isolated orrecombinant antifreeze protein of claim 36, wherein the antifreezeprotein includes the subsequence of amino acids set forth in SEQ IDNO:4.
 41. The isolated or recombinant antifreeze protein of claim 36,wherein the thermal hysteresis activity is greater than 2° C. at aconcentration of about 1 mg/mL.
 42. The isolated or recombinantantifreeze protein of claim 36, wherein the antifreeze protein is YL-4(SEQ ID NO:15).
 43. The isolated or recombinant antifreeze protein ofclaim 36, wherein the antifreeze protein is expressed by a baculovirusvector.
 44. The isolated or recombinant antifreeze protein of claim 36,wherein the antifreeze protein is synthesized by a bacterial cell, afungus cell, a plant cell, or an animal cell.
 45. The isolated orrecombinant antifreeze protein of claim 36, wherein the antifreezeprotein is synthesized by a yeast cell.
 46. The isolated or recombinantantifreeze protein of claim 36, wherein the antifreeze protein issynthesized by an animal cell.
 47. The isolated or recombinantantifreeze protein of claim 36, wherein the nucleic acid encoding theantifreeze protein is synthesized by an insect cell.
 48. The isolated orrecombinant antifreeze protein of claim 36, wherein the antifreezeprotein is derived from Tenebrio sp.
 49. The isolated or recombinantantifreeze protein of claim 44, wherein the antifreeze protein isexpressed externally from the cell.
 50. A liquid comprising arecombinant antifreeze protein, said antifreeze protein comprising thefollowing: (i) a calculated molecular weight of between 7 and 13 kDa;(ii) a thermal hysteresis activity of greater than 1.5° C. at aconcentration of about 1 mg/mL; (iii) the N-terminal amino acid motifset forth in SEQ ID NO:3; (iv) at least 60% amino acid sequence identityto an antifreeze protein YL-4 (SEQ ID NO:15); and (v) at least greaterthan 1 repeat of the 12 contiguous amino acid motif set forth in SEQ IDNO:1.
 51. The liquid of claim 50 wherein said antifreeze proteincomprises at least 5 repeats of the 12 contiguous amino acid motif setforth in SEQ ID NO:1.
 52. The liquid of claim 50 wherein said antifreezeprotein comprises 5 to 12 repeats of the 12 contiguous amino acid motifset forth in SEQ ID NO:1.
 53. The liquid of claim 50, wherein theconcentration of antifreeze protein is between about one part perbillion (1 μg/L) to about one part per thousand (1 g/L).
 54. The liquidof claim 50, wherein the antifreeze protein has a thermal hysteresisactivity greater than 2° C. at a concentration of about 1 mg/mL.